Method for purifying the rabies virus

ABSTRACT

The subject of the invention is a method for purifying the rabies virus, comprising a single ion-exchange chromatography step, said step being cation exchange chromatography according to which:
         a) the supernatant of a culture of cells infected with this virus is brought into contact with a cation exchange chromatography support comprising a polymethacrylate matrix onto which sulfoisobutyl groups have been grafted such that the rabies virus binds to this support, and;   b) the virus is eluted from its support.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication 60/231,394, filed Aug. 5, 2009.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The subject of the invention is a method for purifying the rabies virusthat has been obtained from a culture of cells.

2. Summary of the Related Art

In general, virus harvests obtained from infected cell cultures containnot only the desired viruses, but also the proteins and the DNA of thecells, which are impurities which should be removed. The amounts ofcellular proteins and DNA released are all the greater if the virusesare responsible for considerable cell lysis and/or if the viral harvestsare carried out late. In addition to the impurities of cellular nature,the proteins of the medium containing the infected cells are alsoimpurities that are also intended to be removed during theimplementation of the viral purification process.

When the viruses are used to manufacture vaccines, it is advisable toobtain preparations which are as pure as possible so as to prevent thedevelopment of allergic reactions against the protein impurities. Thereare also countries that limit the maximum authorized amount of cellularDNA in vaccines comprising products obtained from continuous cell linesto 100 pg/vaccine dose or even less.

Various methods for purifying the rabies virus have already beendescribed in the prior art:

U.S. Pat. No. 4,664,912 describes a method for purifying the rabiesvirus by zonal centrifugation after it has been inactivated withβ-propiolactone. Another method consists in combining size exclusionchromatography and anion exchange chromatography when the volume of theharvest of virus to be purified is large.

U.S. Pat. No. 4,725,547 describes a method for purifying the rabiesvirus by affinity chromatography on cellulofine sulfate (a sulfuric acidester of cellulose).

WO 97/06243 describes a method for purifying viruses, in particular therabies virus, from a culture of infected Vero cells. The methodcomprises anion exchange chromatography followed by cation exchangechromatography and is completed by metal-binding affinitychromatography. Using this method, the amount of residual DNA containedin one vaccine dose is ≦30-40 pg, using the “Threshold Total DNA assay”technique.

Kumar A. P et al., in Microbes and Infection (2005) 7; 1110-1116, havecompared two methods for purifying the rabies virus from a culturesupernatant of infected Vero cells that were cultured in a mediumcontaining fetal calf serum. It shows that the purification method basedon the use of an anion exchange chromatography column (DEAE-sepharoseCL-6B) is more effective than the method of purification by zonalcentrifugation on a sucrose gradient, since, at a comparable degree ofpurity in terms of amount of residual proteins and nucleic acids, therabies virus yield is better with the chromatographic method.Frazatti-Gallina N. M., in Vaccine (2004) 23; 511-517, has alsodescribed a method for purifying the rabies virus from a culturesupernatant of infected Vero cells that were cultured in a medium freeof fetal calf serum. The purification method also implements a step ofanion exchange chromatography on a DEAE-cellulose-based support, after astep of clarifying and concentrating the viral harvest. Using thismethod, the amount of residual DNA measured by slot/blot hybridizationtechnique is <23 pg per vaccine dose.

Thus, when the method for purifying the rabies virus comprises an ionexchange chromatography step, there is virtually systematically an anionexchange chromatography step so as to retain the nucleic acids of themedium containing the virus to be purified, on the chromatographicsupport.

SUMMARY OF THE INVENTION

A particularly effective virus purification method should make itpossible to optimally remove the protein impurities and the cellular DNAwhile at the same time guaranteeing a maximum yield of purified rabiesvirus, and there still exists the need to find new methods which meetthese requirements.

One objective of the invention is to provide a new purification methodwhich meets these requirements.

Another objective of the invention is to provide a purification methodwhich is suitable for purifying the rabies virus from a harvest which isfree of animal serum or of serum protein and in particular to provide apurification method in which every step is carried out by means ofproducts of non animal origin.

To this effect, a subject of the present invention is:

A method for purifying the rabies virus, comprising a singleion-exchange chromatography step, said step being cation exchangechromatography according to which:

-   -   a. the supernatant of a culture of cells infected with this        virus is brought into contact with a cation exchange        chromatography support comprising a polymethacrylate matrix onto        which sulfoisobutyl groups are grafted by covalent bonding such        that the rabies virus binds to this support, and, secondly;    -   b. the virus is eluted from its support.

According to one aspect of the method according to the invention, theculture supernatant of cells infected with the rabies virus is free ofanimal serum or free of any serum protein.

According to another aspect, the culture supernatant of cells infectedwith the rabies virus is free of any exogenous protein of animal origin.

According to a still another aspect, the culture supernatant of cellsinfected with the rabies virus, which is free of any exogenous proteinof animal origin, contains exogenous proteins of non animal origin at aconcentration ≦15 mg/l.

According to another aspect, the culture supernatant of cells infectedwith the rabies virus is free of any exogenous product of animal origin.

According to a still another aspect, the culture supernatant of cellsinfected with the rabies virus does not contain proteins having amolecular weight above 10 kDa.

In one embodiment of the method according to the invention, the culturesupernatant of cells infected with the rabies virus is clarified beforeit is brought into contact with the cation exchange chromatographysupport.

Typically, the purification method according to the invention ischaracterized in that the amount of virus measured in the eluatecorresponds to at least 70%, and preferably to at least 80% of theamount of virus measured in the supernatant that was brought intocontact with the chromatographic support.

More particularly, the purification method is characterized in that theamount of total proteins measured in the eluate corresponds to less than40% of the amount of total proteins measured in the supernatant that wasbrought into contact with the chromatographic support and in that theamount of DNA measured in the eluate corresponds to less than 5%,preferably to less than 2.5%, and even more preferably to less than 1%of the amount of DNA measured in the supernatant that was brought intocontact with the chromatographic support.

In another embodiment of the method according to the invention, afterhaving eluted the virus from its chromatographic support, the eluate isoptionally concentrated and then treated with a nuclease.

In one particular aspect, the nuclease is an endonuclease.

In another embodiment of the invention, the method comprises anadditional step of purifying the rabies virus, according to which theeluate treated with a nuclease is then subjected to anultracentrifugation on a sucrose gradient and the fraction(s) of thegradient which contain(s) the purified virus is (are) recovered.

In another aspect of the invention, the purified rabies virus is theninactivated by means of a viral inactivation agent.

In one particular aspect, the viral inactivation agent isβ-propiolactone.

In a further aspect, all the steps of the method according to theinvention are carried out by means of products of non animal origin.

A subject of the invention is also a method for the manufacture of arabies vaccine, according to which:

-   -   a) a culture of cells is infected with the rabies virus,    -   b) the rabies virus is purified from the infected-cell culture        supernatant that is recovered at the end of step a) according to        a method of the invention,    -   c) the suspension of purified virus obtained in b) is mixed into        a storage buffer, and,    -   d) the suspension of purified virus obtained in c) is divided up        in the form of single-dose or multidose vaccines.

According to another aspect, it is a method for the manufacture of arabies vaccine, according to which:

-   -   a) a culture of cells is infected with the rabies virus,    -   b) the rabies virus is purified from the infected-cell culture        supernatant that is recovered at the end of step a) according to        a method of the invention,    -   c) the suspension of purified virus obtained in b) is mixed into        a lyophilization buffer,    -   d) the mixture obtained in c) is divided up in the form of        single-dose or multidose vaccines, and    -   e) the vaccine doses are lyophilized.

Finally, a subject of the invention is a vaccine containing purified andinactivated rabies virus, characterized in that the amount of residualDNA measured by quantitative PCR and the amount of total proteins whichare present in one effective dose of vaccine (or in one dose of vaccinewhich contains 2.5 IU determined according to European Pharmacopeiamonograph 0216 (“Rabies Vaccine for Human Use Prepared in CellCultures”)) are, respectively, less than 20 pg and less than 40 μg, andpreferably less than 10 pg and less than 20 μg.

Preferably, at least 70% of the total proteins that are present in oneeffective dose of vaccine are rabies virus proteins.

More preferably the vaccine is free of any exogenous product of animalorigin.

DETAILED DESCRIPTION OF THE INVENTION

The method for purifying the rabies virus according to the inventioncomprises a single ion exchange chromatography step, saidchromatographic step being a cation exchange chromatography. In thescope of the present invention, it is however well understood that themethod of purifying rabies virus is not limited to only onechromatography step, but may include one or several additional steps,provided that the additional steps are not ion exchange chromatography.Contrary to what the prior art leads one to envision, the inventors havedemonstrated that the performance levels of a cation exchangechromatography, the support of which comprises a polymethacrylate matrixonto which sulfoisobutyl are grafted by covalent bonding are better thanthose of an anion exchange chromatography. Without wishing to be boundby theory, it seems that the rabies virus has a double, positive andnegative, polarity which enables it to bind both to anion exchangesupports (of the DEAF type) and to a strong cation exchange supports (ofthe SO₃ ⁻ type). For the purpose of the invention, the “performancelevels of a chromatography” are calculated on the basis of the amount ofrabies virus and of DNA that are found in the eluate compared with theinitial amount of virus and of DNA that were brought into contact withthe chromatographic support, within the loading capacity limits of thechromatographic support. At equivalent amounts of virus and of DNA thatwere brought into contact with the chromatographic support, the higherthe amount of virus found in the eluate and the smaller the amount ofresidual DNA, the better the performance level of the chromatographicsupport. Sulfoisobutyl groups are grafted onto the polymethacrylatematrix through flexible polymeric chains which facilitate theinteraction between the rabies virus and the cation exchanger.Preferably, the polymeric chain is made of a chain of monomer unitshaving the following formula:

These polymeric chains are obtained by graft polymerization of a monomerhaving the following formula CH₂═CH—CO—NH—C(CH₃)₂—CH₂—SO₃ ⁻ onto thepolymethacrylate matrix-based support in presence of a cerium-basedcatalyst. The mean length of the polymeric chains is generally between15 and 25 monomer units. The chromatographic support is conventionallyin the form of particles, the size of which generally ranges between 20and 100 μm, preferably between 40 and 90 This type of support is inparticular sold by MERCK under the name Fractogel® EMD SO3⁻ (type M).This type of support is placed in a chromatography column, the lengthand diameter of which are chosen according to the volume of harvest tobe purified. The results presented in example 1 show that theperformance levels of the tests carried out on anion exchangerchromatographic supports and on cation exchanger chromatographicsupports are mediocre from the viewpoint of those that can be obtainedwith the Fractogel® EMD SO₃ ⁻ support. Surprisingly, except for thechromatography carried out on a Fractogel® EMD SO₃ ⁻ support, the virusyields obtained with all the other cation exchanger chromatographicsupports tested are very low (less than 10% of the amount of virusbrought into contact with the support is found in the eluate). The virusyields obtained with anion exchanger chromatographic supports are better(between 40 and 70%) but are nevertheless lower than the virus yieldobtained with a Fractogel® EMD SO₃ ⁻ support, which is higher than 70%and generally higher than 80%. Therefore, the performance levels areparticularly good when a strong cation exchanger chromatographic supportcomprising a polymethacrylate matrix onto which sulfoisobutyl groups(which play the role of ligands) are grafted by covalent bonding isused. A ready-for-use macroporous charged membrane, where thesulfoisobutyl groups are grafted onto a polymethacrylate membrane, mayalso be used as support.

Both more than 60% of the total proteins, and at least 2.5 log₁₀ of DNA,preferably at least 3.0 log₁₀ of DNA and even more preferably at least3.5 log₁₀ of DNA (which corresponds to the removal of more than 99% ofthe cellular DNA) are removed in a single cation-exchange chromatographystep according to which the support comprises a polymethacrylate matrixonto which sulfoisobutyl groups are grafted by covalent bonding, whileat the same time conserving a rabies virus yield of at least 70% (i.e.there is in the eluate an amount of rabies virus which corresponds to atleast 70% of that which was initially brought into contact with thechromatographic support). These performance levels are observed inparticular when the culture supernatant of cells infected with therabies virus does not contain animal serum or serum protein (whichcorresponds to any protein component of the serum, like albumin).Therefore, the amounts of total proteins and of DNA measured in thechromatographic eluate contains, respectively, less than 40% of theamount of total proteins present in the volume of culture supernatant ofcells infected with the rabies virus and less than 5%, preferably lessthan 2.5%, and even more preferably less than 1% of the amount of DNAalso present in the volume of supernatant that was brought into contactwith the chromatographic support. On the other hand, at least 70% of theamount of rabies virus present in the volume of supernatant that wasbrought into contact with the chromatographic support is found in theeluate.

For the purpose of the invention, the “total proteins” means all theproteins that are present in the material that is analyzed. They arerepresented by the rabies virus proteins, the cellular proteins, theproteins of the cell culture medium and of the viral infection mediumand also possibly by the proteins that are possibly introduced duringthe purification process (for instance benzonase). The cellularproteins, the proteins of the culture medium and of the viral infectionmedium as well as the proteins that are possibly introduced during thepurification process are the impurities (protein impurities) that areintended to be removed, whereas the intention is to conserve as much aspossible the proteins of the rabies virus. The rabies virus proteins areglycoprotein G, nucleoprotein N, Phosphoprotein P, matrix protein M, andRNA dependant RNA polymerase L.

The amount of rabies virus is evaluated on the basis of the measurementof the rabies virus glycoprotein G under a form correctly folded. Thisis usually carried out by means of a “sandwich” ELISA method with theuse of two antibodies that recognizes at least one, or preferably twoconformational epitopes of the glycoprotein G, as it is described inexample 1. A rabies virus neutralizing antibody recognizing aconformational epitope located on the antigenic site II of glycoproteinG (Journal of Clinical investigation (1989), vol. 84, pp. 971 to 975) isused as capture antibody, and a rabies-virus-neutralizing antibodyrecognizing a conformational epitope located on the antigenic site IIIof glycoprotein G (Biologicals (2003), vol. 31, pp. 9 to 16) is used asrevealing antibody. The results are expressed in IU on the basis of theuse of a reference standard which has been calibrated with respect tothe NIBSC international reference.

The Bradford method, which is well known to those skilled in the art, isused to measure the amount of total proteins.

To measure the amount of DNA, a quantitative PCR (qPCR) method based onthe quantification of a DNA fragment of the cell genome is preferablyused (a DNA fragment which is repeated many times in the cell genome ispreferably targeted). When the rabies virus is produced from Vero cells,the quantification of the residual DNA during the virus purificationprocess is based on the quantification of the African green monkeyalpha-satellite DNA fragment after PCR amplification using a methodsimilar to that which is described by Lebron J. A. et al., inDevelopments in Biologicals (2006), vol 123, pp. □135-44, and details ofwhich are given in example 1. This method is very advantageous since itmakes it possible to measure all the cellular DNAs which have more than200 base pairs.

The culture supernatant containing the rabies virus is produced from acell culture that has been infected with the rabies virus. Any cellculture in which the rabies virus replicates is suitable for the subjectof the invention. These cultures may be primary cultures of animaltissues, such as primary chicken embryo cultures (for example, primarychicken embryo fibroblast (PCEF) cultures), primary cultures of neonatemouse brains, or primary monkey, rabbit, hamster or dog kidney cultures,but a cell culture originating from established cell lines that derivefrom primary cultures of animal tissues is preferably used. The linesare in particular cell lines originating from primates, such as theVERO, WI-38, MRC5, PERC.6 line or the 293 line, from horses, from cows(such as the MDBK line), from sheep, from dogs (such as the MDCK line),from cats or from rodents (such as the BHK21, HKCC or CHO line).Particularly preferably, the Vero cell line is used, said line havingmany advantages: it is a continuous line that can be readily cultured onthe industrial scale, which has a very weak mutagenic capacity and whichis very sensitive to the rabies virus.

The cells can be cultured in suspension or on a support depending onwhether or not they have adhesion properties, in a batch or a fed batchmode or according to a continuous perfusion culture mode. In the case ofcell line culture on the industrial or semi-industrial scale,biogenerators are generally used, the volume of which is greater than 10liters and may go up to more than 2000 liters, comprising a stirringsystem, a device for injecting a CO₂ gas stream and an oxygenationdevice. They are equipped with probes which measure the internalparameters of the biogenerator, such as the pH, the dissolved oxygen,the temperature, the pressure of the tank, or certain physicochemicalparameters of the culture (such as the consumption of glucose or ofglutamine or the production of lactates and of ammonium ions). The pH,oxygen and temperature probes are connected to a bioprocessor whichcontinually regulates these parameters. In the case of adherent celllines cultured in biogenerators, the culture medium containsmicrocarriers which are microbeads to which the cells attach. Thesemicrocarriers are kept in suspension by mechanical stirring, or by meansof a gas stream. In the case of the Vero cell line, use is normally madeof microcarriers, of which the adhesive electrostatic matrix is based ondextran substituted with N,N-diethylaminoethyl groups, which are sold inparticular by Amersham Biosciences under the name Cytodex 1 or Cytodex2. Cytodex 3 microbeads sold by Amersham Biosciences may also be used asmicrocarriers.

The medium which is used for culturing the cells, also known as cellculture medium, may or may not be supplemented with serum of animalorigin, or contain one or more serum proteins such as human albumin, orbe free of any protein of animal origin, or even be free of any protein.Usually, a cell culture medium free of animal serum or even free of anyserum protein like albumin that may be responsible for the developmentof a hypersensitivity reaction in the vaccinated individual is used(Swanson M. C. et al., Journal of Infectious Disease (1987);155(5):909-913). Preferably, a culture medium free of any protein ofanimal origin or even better free of any product of animal origin isused. The term “protein or product of animal origin” means a protein ora product, of which the manufacturing process comprises one or severalsteps in which a material originating from animals or humans is used.This makes it possible to decrease the risks of transmission of diseasessuch as BSE which may be linked to the use of biological products ofanimal origin. The culture media contain generally small amounts ofproteins in the form of recombinant proteins or proteins extracted fromplants (soya, rice, etc. . . . ). They commonly contain lowmolecular-weight proteins (≦10 kDa) at low concentrations. Usually, theconcentration of total proteins in these media is ≦15 mg/l measured bythe Bradford method. This is the case in particular of the VP SFM mediumsold by Invitrogen, which is suitable for the process according to theinvention, in particular for cultivating Vero cells. Mention is alsomade of the media Opti Pro™ serum-free (InVitrogen), Episerf(InVitrogen), Ex-cell® MDCK (Sigma-Aldrich), Ex-Cell™ Vero (SAFCbiosciences), MP-BHK® serum free (MP Biomedicals), SFC-10 BHK expressserum free (Promo cell), SFC-20 BHK express protein free (Promo cell),HyQ PF Vero (Hyclone Ref. SH30352.02), Hyclone SFM4 Megavir, MDSS2medium (Axcell biotechnology), Iscove's modified DMEM medium (Hyclone),Ham's nutrient media (Ham's-F10, Ham's-F12), Leibovitz L-15 medium(Hyclone), which are free of any product of animal origin and whichcontain low amounts of proteins (≦15 mg/l).

The rabies virus may come from any origin provided that it reproduces inrabies virus-sensitive cells. Rabies virus strains which have beenestablished from primary isolates are generally used, such as thePasteur 2061 strain, the VP-11 strain or the Pitman-Moore 1.503-3 Mstrain. These very virulent strains are intended for the manufacture ofinactivated rabies vaccines. Use may also be made of rabies virusstrains that have been attenuated for the purpose of producing a liveattenuated rabies vaccine. They are, for example the SAD Bern strain orthe SAD B19 strain or strains derived therefrom, such as the SAG1 andSAG2 strains that derive from the SAD Bern strain owing to the existenceof point mutations in the rabies virus glycoprotein G (EP 350398, EP583998). EP 1253 197 also describes other, more stable, attenuatedrabies virus strains, which comprise one or more point mutations in therabies virus phosphoprotein P and glycoprotein G.

The culture supernatant of the cells infected with the rabies virus isobtained using a viral infection medium, which replaces the cell culturemedium at the time of the infection of the cells and which serves forthe production of the rabies virus by the infected cells. The chemicalcomposition of the viral infection medium may be identical to or may bedifferent than the composition of the cell culture medium. The viralinfection medium, like the cell culture medium, may be supplemented withserum of animal origin or with one or more serum proteins, but a viralinfection medium free of animal serum or free of any serum protein ispreferably used. Preferably, a viral infection medium free of anyprotein of animal origin or even better free of any product of animalorigin is used to decrease the risks of transmission of diseases such asBSE which may be linked to the use of biological products of animalorigin. It contains generally small amounts of proteins in the form ofrecombinant proteins or proteins extracted from plants (soya, rice, etc.. . . ). Commonly, it contains low molecular-weight proteins (≦10 kDa)at low concentrations. Usually, the concentration of total proteins insuch medium is ≦15 mg/l measured by the Bradford method. As medium freeof any product of animal origin and that contains low amounts ofproteins (≦15 mg/l), mention is made of VP SFM medium (Invitrogen),Leibovitz L-15 medium, MEM medium (Sigma-Aldrich), Hyclone SFM4 Megavir™medium (Hyclone) or, optionally, one of the media based on plantextracts as described in application WO 99/4768. For example, when therabies virus is produced from a Vero cell culture, the infected-cellculture supernatant is generally obtained by using a medium free of anyserum protein or even free of any product of animal origin. Depending onthe composition of the cell culture medium and of the viral infectionmedium, the infected-cell culture supernatant may advantageously be:

-   -   free of any serum protein, and/or    -   free of any exogenous protein of animal origin, and/or    -   free of any exogenous product of animal origin.

When the infected-cell culture supernatant is free of any exogenousprotein of animal origin or is free of any exogenous product of animalorigin, the exogenous proteins of non animal origin that are present inthe supernatant are preferably at a low concentration (≦15 mg/l)measured by the Bradford method.

The term “exogenous protein or exogenous product” means any protein orproduct that is not a component of the rabies virus or of the cells usedfor the production of rabies virus. Exogenous proteins or exogenousproducts are components that are introduced during the production and/orthe purification of rabies virus. For instance, the proteins or theproducts that are optionally present in the medium composition, theenzymes such as trypsin or benzonase that are introduced during theproduction and/or the purification of rabies virus are exogenousproteins or exogenous products. The exogenous proteins/products are ofanimal origin when their manufacturing process comprises one or severalsteps, in which a material originating from an animal or from a human isused. The exogenous proteins/products are of non animal origin when theyare produced by other means, for example by using a plant material, bychemical synthesis or by genetic recombination using yeasts, bacteria orplants.

The harvesting of the culture supernatants containing the infectiousvirus generally begins 2 to 3 days after having infected the cells, andcontinues for about two weeks since the virus is not very lytic. Aftereach harvest, the infected cells are put back in culture with new viralinfection medium. The proteins that are found in the culturesupernatants are of cellular and viral origin but also originate fromthe viral infection medium and to a minimal extent originate from thecell culture medium. It is possible to store all the harvests, or tokeep only the harvests which have very high infectious titers. Theharvests can be stored individually. It is also possible to mix severalharvests. The harvests are normally kept at a temperature about +5° C.or in frozen form.

As a precaution, the culture supernatant containing the virus to bepurified is often clarified before the cation exchange chromatographystep so as to remove the coarse cell debris, the aggregates possiblypresent, and the residual microcarriers when the cell culture has beencarried out on microcarriers. Any clarification method well known tothose skilled in the art may be used in the method according to theinvention. It is possible, for example, to centrifuge the supernatantsor to perform size exclusion chromatography in order to clarify theculture supernatant. The clarification is usually carried out bytangential and/or frontal membrane filtration and/or depth filtrationusing one or more filters, the porosity of which usually ranges between0.2 and 1.5 μm, preferably between 0.4 and 1.0 μm. The clarification canbe carried out by means of a single filtration of the culturesupernatant in cases where there is little debris and few aggregates.The clarification can also be carried out by combining at least twosuccessive filtrations, the first filtration being carried out, forexample, with a relatively coarse filter having, for example, a porosityof between 0.3 and 1.5 μm, while the second filtration is carried outwith a relatively fine filter having, for example, a porosity of between0.2 and 0.5 μm. It is also possible to provide for a prefiltration step,beforehand, using a prefilter with a large porosity (between 2 and 10μm) in order to remove the large debris. As filters that are suitablefor the object of the invention, mention may be made of cellulosefilters, regenerated cellulose fibers, cellulose fibers combined withinorganic filters (for example, based on diatoms, on perlite or on fumedsilica), cellulose filters combined with inorganic filters or organicresins, or polymer-based filters, such as, for example, nylon,polypropylene, fluorinated polyvinylidene or polyethersulfone filters.These filters are in particular sold under the name Durapore®, Millipak®or Millidisk™ distributed by the company Millipore or the filtersdistributed by the company Pall. As depth filter system, mention may bemade of the depth filters of the AP series (AP01), of the CP series(CP10, CP30, CP50, CP60, CP70, CP90), of the HP series (HP10, HP30,HP50, HP60, HP70, HP90), of the CA series (CA10, CA30, CA50, CA60, CA70,CA90) and of the SP series (SP10, SP30, SP50, SP60, SP70, SP90), whichare supplied by the company CUNO, the CUNO Delipid and Delipid Plusfilters, and the depth filters of the CE series (CE15, CE20, CE25, CE30,CE35, CE40, CE45, CE50, CE70, CE75) and of the DE series (DE25, DE30,DE35, DE40, DE45, DE50, DE55, DE60, DE65, DE70, DE75), which aresupplied by the company Millipore corp., the filters of the HC series(A1 HC, B1HC, COHC) and the Clarigard and Polygard filters from thecompany Millipore corp. As another filtration system, mention may alsobe made of the depth filters distributed by ManCel associates (forexample, PR 12 UP, PH12, PR 5 UP) or the filters from PALL or from thecompany SeitzShenk Inc (for example, Bio20, SUPRA EKIP, KS-50P).Advantageously, the clarification step may be carried out by means of afilter capsule comprising two membranes with different porosities, whichamounts to carrying out two successive filtrations in a single step. TheSartopore-2 filter capsules sold by Sartorius, comprising a 0.8 μmmembrane and a 0.45 μm membrane, are very suitable for clarifying theculture supernatant. A prefiltration step as indicated above may also beincorporated beforehand, as required.

Irrespective of whether or not the culture supernatant is clarified, itis if required adjusted to a pH of between 7 and 8, preferably between7.0 and 7.6. It is also verified that the conductivity is ≦20 mS/cm,preferably between 10 and 20 mS/cm, and particularly preferably between14 and 18 mS/cm. The culture supernatant is then introduced into acation exchange chromatography column, the support of which isconstituted of particles of Fractogel® EMD SO3⁻ (type M) and thedimensions of which are suitable for the volume of supernatant to bepurified. The column is pre-equilibrated in a low-ionic-strengthequilibration buffer (≦200 mM). The column is then washed with a washingbuffer which generally has the same composition as the equilibrationbuffer. What leaves the column is mainly the protein impurities and thenucleic acids. The virus which has been retained on the column is theneluted using an elution buffer, the ionic strength of which is at least400 mM. By way of example of buffer solutions suitable for the object ofthe invention, mention may be made of 20 mM Tris buffer containing 150mM NaCl, pH=7.5, for equilibrating and washing the column, and 20 mMTris buffer containing 600 mM NaCl, pH=7.5, as virus elution buffer, butany other equivalent buffer solution could also be suitable. The loadingcapacities of the column being respected, more than 70% of the amount ofvirus that was initially introduced in the column is thus recovered inthe eluate, while the amounts of DNA and of total proteins recovered inthe eluate represent, respectively, less than 5% and less than 40% ofthe initial amounts of DNA and of total proteins that were initiallyintroduced in the column. These results are obtained in particular whenthe infectious culture supernatants do not contain serum of animalorigin or do not contain serum protein or contain exogenous proteins ofnon animal origin at low concentration (≦15 mg/l).

The inventors have also shown that, by combining a cation exchangechromatography step on a support comprising a polymethacrylate matrixonto which sulfoisobutyl groups are grafted with an enzymatic nucleicacid digestion step, it is also possible to reduce the level of residualDNA by approximately a further 1.5 to 2 Log₁₀, to such an extent that,by combining these two steps, at least 4.0 log₁₀ of DNA are successfullyremoved. By way of comparison, using a method for removing nucleic acidsbased on an enzymatic nucleic acid digestion that is repeated twice, alevel of DNA removal which does not exceed 3.5 log₁₀ is obtained, thesecond digestion not enabling the residual level of DNA to be reducedbeyond a further 0.5 to 1 Log₁₀ (see example 2). As enzymatic agent fornucleic acid digestion, use may be made of one or more enzymes,preferably an RNAse and/or a DNAse, or a mixture of endonucleases knownto those skilled in the art, for instance Pulmozyme™. In the context ofthe method according to the invention, Benzonase™, preferably obtainedby genetic recombination, is generally used, in a concentration range ofgenerally between 1 and 50 U/ml. It is an endonuclease which acts byrapidly cleaving cellular DNA and ARN and which reduces the viscosity ofthe medium. The temperature and the duration of the enzymatic treatmentare parameters that can be easily controlled by those skilled in the artand that depend on the initial concentration of the endonuclease in thereaction medium. To prevent any aggregation phenomenon, a very smallamount of a surfactant, which is preferably nonionic, such as poloxamer188 (Pluronic F 68) can be added, at a very low concentration, to thechromatographic eluate. Prior to the enzymatic treatment step, theeluate may optionally be concentrated when the volume is large. Theconcentration step is generally carried out by ultrafiltration on amembrane, having a cutoff threshold of between 100 kDa and 300 kDa,preferably between 100 kDa and 200 kDa. Ultrafiltration is characterizedby a tangential flow over the membrane which induces a force thatenables the molecules to diffuse through the porous membrane. The flowimposed by a recirculation pump is divided up into two components: therecirculation flow which performs the sweep (or retentate flow) and thefiltrate (permeate) flow which passes through the membrane. Thecomposition of the membrane may, in a nonlimiting manner, be made ofregenerated cellulose, of polyethersulfone, of polysulfone or ofderivatives of these products. It may be in the form of flat sheetsinside cassettes (in particular for tangential ultrafiltration) or ofhollow fibers. The membranes are in particular sold by Pall under thename Omega™, by Millipore under the name Biomax™ membranes and bySartorius under the name Sartocon®. A back-pressure may also be appliedon the filtrate (permeate) side in order to reduce the transmembranepressure, as is described in WO 2006/108707. As the eluate passesthrough the membrane, the volume of the eluate decreases and the virusthat does not pass through the membrane is concentrated. When the eluateis ultrafiltered, the volume thereof can be decreased by a factor thatmay range from 1 to 100, or even 150, thus making it possible to obtainthe desired final volume. This concentration step may be completed witha diafiltration step which makes it possible to modify the compositionof the buffer without however modifying the volume of the retentate.This is recommended when the composition of the buffer in the retentate,owing to the decrease in volume of the eluate, is no longer compatiblewith good enzymatic activity of the endonuclease. A buffer, thecomposition of which is compatible with good enzymatic activity of theendonuclease, is then added to the retentate recirculation flow. By wayof example, and without being restrictive in nature, mention is made ofbuffer compositions compatible with good enzymatic activity of anendonuclease such as Benzonase™: they contain a Tris buffer in aconcentration range by molarity of from 10 to 50 mM, MgCl₂ in aconcentration range of generally from 1 to 10 mM and, optionally,another salt, such as NaCl, in a concentration range of from 100 mM to600 mM, the pH of these buffer solutions being in a pH range of from 7.0to 8.0. As indicated above, the eluate recovered after thechromatographic step may also be treated directly with an endonuclease,such as Benzonase™ by adding MgCl₂ in a concentration range of generallyfrom 1 to 10 mM before the addition of Benzonase.

In order to complete the removal of the protein impurities, and inparticular the removal of Benzonase, the eluate which has been treatedwith an endonuclease is subjected to a sucrose-gradientultracentrifugation step, it being possible for this ultracentrifugationstep to be repeated one or more times. The ultracentrifugation enablesthe rabies virus to be isolated by adjusting the difference insedimentation coefficient between the various entities present in theeluate: the protein impurities migrate into the low sucrose densities(≦35% of sucrose), whereas the rabies virus is in the higher sucrosedensities (≧35% of sucrose). The sucrose-gradient ultracentrifugationstep is generally carried out according to the “continuous flow” methodwhen the volumes to be treated are large or in a rotor when the volumesare relatively small. The sucrose-gradient ultracentrifugation isusually carried out on “sucrose cushions” at a temperature about 5° C.The eluate which has been treated with an endonuclease may be in anonconcentrated form or in a form concentrated 10 times, 20 times, 50times, 100 times, 150 times, or even more. The ratio between the totalvolume of the eluate to be ultracentrifugated and the total volume ofthe sucrose gradient is usually between 0.5 and 2.0, and preferablybetween 0.5 and 1.5. The density of the low-density sucrose “cushion” isusually within a range of from 10% to 35% w/w, while the density of thehigh-density sucrose “cushion” is usually within a range of from 40% to60% w/w. The volume of low-density sucrose is generally greater than thevolume of high-density sucrose, all the more so if the volume of theeluate subjected to the ultracentrifugation step is large. The ratiobetween the volume of high-density sucrose and the volume of low-densitysucrose is usually between 0.3 and 0.7. The duration of theultracentrifugation depends on the ultracentrifugation speed, which isin general ≧30000 g. An ultracentrifugation time of 2 hours is generallysufficient when the ultracentrifugation speed is ≧65000 g. Afterultracentrifugation, the product distributed within the gradient istaken off using a peristaltic pump and fractionated. The amount ofsucrose contained in each fraction is measured using a refractometer. Ineach fraction, the amount of rabies virus is measured, on the basis ofassaying the glycoprotein gpG by ELISA, as well as the amount of totalproteins by the Bradford method. The separation profile is thusestablished. At the end of these analyses, the fractions of the gradientcontaining essentially the rabies virus are selected and combined.During this step, more than 85% and preferably more than 90% of thetotal proteins are removed, this being essentially protein impuritiessince the rabies virus yield is ≧70% and preferably ≧80%.

Thus, when the step of clarification of the infected-cell culturesupernatant, the step of cation exchange chromatography on a supportcomprising a polymethacrylate matrix onto which sulfoisobutyl groups aregrafted by covalent bonding, optionally followed by a concentrationand/or diafiltration step, the step of treatment with an endonucleaseand the step of sucrose-gradient ultracentrifugation are combined in thepurification method according to the invention, a composition ofpurified rabies is obtained which, for an amount of rabies viruscorresponding to 4.5 IU (on the basis of measuring gpG by ELISA) or toone effective dose of vaccine, contains less than 50 pg of residual DNA,preferably less than 20 pg of residual DNA and even more preferably lessthan 10 pg of residual DNA (measured by qPCR) and contains less than 40μg of total proteins, preferably less than 20 μg of total proteins(measured by the Bradford method). Usually, at least 70% of the totalproteins are viral proteins. The overall yield of purified virus is atleast 45%, and preferably at least 50%, and even more preferably atleast 60%. The overall yield of purified virus is calculated on thebasis of the ratio of the amount of virus present in the gradientfraction(s) recovered after ultracentrifugation to the amount of virusinitially present in the volume of infected-cell culture supernatant tobe purified. Preferably, the rabies virus infected-cell culturesupernatant is free of any serum protein, or free of any exogenousprotein of animal origin, or even free of any exogenous product ofanimal origin to warrant in particular a better biological safety of thevaccine composition.

By way of example, table I below indicates, for each step of the methodfor purifying the rabies virus from a culture supernatant of infectedVero cells, the amounts of virus recovered and also the amounts of DNAand of proteins removed.

TABLE I Table summarizing the various steps of the rabies viruspurification process with the associated virus yields and the amounts ofDNA and of proteins removed. Amount Amount of Amount of virus proteinsof DNA recovered removed removed (as %)* (as %)** (en log₁₀)*** in thein the in the Step step overall step overall step overall Clarification90 90 ND ND ND ND Chromatography on 89 80 60 60 ND ND EMD/SO₃ ⁻ supportUltrafiltration/ 97 77 18 67 2.7 2.7 diafiltration Benzonase treatment +70 54 88 96 1.6 4.3 sucrose-cushion ultracentrifugation *the amount ofrabies virus is determined on the basis of assaying the rabies virusglycoprotein G by ELISA according to the method described in example 1.**the amount of total proteins is measured by the Bradford technique.***the amount of DNA is measured by qPCR according to the methoddescribed in example 1. ND: Not determined

The purified rabies virus recovered after the ultracentrifugation stepis generally in a form that is too highly concentrated and that is oftendiluted so as to prevent the formation of viral aggregates duringstorage. A phosphate buffer at a pH of approximately 8, optionallysupplemented with a saline solution, for instance a sodium chloridesolution, is normally used as dilution buffer. Thanks to the processaccording to the invention, the suspension of purified rabies virusobtained can be used as live or attenuated virus vaccine or asinactivated virus vaccine. When the vaccine is intended for humanmedicine, the suspension of purified rabies virus is generallyinactivated.

Viral Inactivation Step

The method for purifying the rabies virus as described in the inventionends with a viral inactivation step when the virus is intended for themanufacture of an inactivated vaccine. The viral inactivation can becarried out by means of chemical agents well known to those skilled inthe art, such as formaldehyde, glutaraldehyde or β-propiolactone. It isalso possible to use the inactivation method as described in WO2005/093049, which consists in bringing the purified viral solution intocontact with a photoactivatable hydrophobic compound and in exposingthis mixture to light. Among the photoactivatable hydrophobic compounds,mention is made of azidobenzene, 1-azidonaphthalene,4-azido-2-nitro-1-(phenylthio)benzene, 1-azido-4-iodobenzene,1-azido-5-iodonaphthalene, 3-phenyl-3H-diazirene,3-phenyl-3-(trifluoromethyl)-3H-diazirene,3-(3-iodophenyl)-3-(trifluoromethyl)-3H-diazirene, 1-azidopyrene,adamantine diazirene, 12-(4-azido-2-nitrophenoxy)stearic acid,w-(m-diazirinophenoxy) fatty acid, 12-[(azidocarbonyl)oxy]stearic acid,12-azidostearic acid, 11-(3-azidophenoxy)undecanoic acid orw-(m-diazirinophenoxy)undecanoic acid or 1,5-iodonaphtyl azide.Preferably, β-propiolactone (BPL) is used because it is both a viralinactivation agent and an alkylating agent resulting in cleavages in theDNA. It may therefore also contribute to reducing the level of residualDNA and to inhibiting its biological activity. The inactivation of therabies virus is carried out by means of a solution of β-propiolactonediluted to between 1/3500 and 1/4000 (final volume concentration in thesolution containing the purified virus) at a temperature ofapproximately 12° C. The lower the concentration of β-propiolactone, thelonger the time necessary for inactivation of the virus. Generally, theinactivation of the virus is carried out in a time period ranging from12 h to 48 h. The activity of the β-propiolactone is neutralized bysimply heating the solution at a temperature of approximately 37° C. forapproximately 2 h. It is then verified that the pH of the solutionis >7.0. The pH is rectified, if necessary, by means of a dilutesolution of sodium hydroxide, or alternatively, before the treatmentwith β-propiolactone, the solution containing the purified virus isbuffered with a solution based on a phosphate buffer pH ˜8, therebypreventing any acidification of the solution during the hydrolysis ofthe β-propiolactone.

The viral suspension purified according to the method of the inventionis in general stored in a storage buffer such as, for example, a Trisbuffer or a phosphate buffer at a pH around 8.0. Although the purifiedviral suspension can be mixed directly with the storage buffer, a stepof diafiltration by ultrafiltration into the storage buffer is generallycarried out, completed if necessary by a step of concentration if thepurified virus is not sufficiently concentrated. In case anultrafiltration step is carried out, it is advisable to use a porousmembrane having a cut-off threshold of between 5 kDa and 100 kDa,preferably between 8 kDa and 50 kDa. The stock of purified virus thusprepared is finally sterilized by filtration through a membrane having aporosity ≦0.2 μm, and then stored at approximately +5° C., preferably ina frozen form before being divided up in the form of single-dose ormultidose vaccines. An additional step of lyophilization of the vaccinepreparations may also be included. In this case, the storage buffercomposition is chosen in such a way that it can be lyophilized. Theoverall yield of inactivated purified virus, calculated here on thebasis of the ratio of the amount of virus present in the stock ofpurified virus obtained after the ultimate sterilizing filtration stepto the amount of virus initially present in the volume of theinfected-cell culture supernatant to be purified is still at least 40%,which attests to the industrial advantage of such a method ofpurification.

Ultimately, a vaccine containing purified rabies virus may be producedwith a very good yield and with a very high level of purity using aprocess according to which the steps of:

-   -   producing a cell batch from a cell bank, usually from a working        cell bank, and of,    -   producing rabies virus after infection of the cell batch with a        viral seed lot, and of,    -   purifying rabies virus from the infected-cell culture        supernatant are all carried out with the use of exogenous        products of non-animal origin.

Thanks to the process of the invention a rabies vaccine may be obtainedfree of any exogenous product of animal origin.

Accordingly, a subject of the invention is also a process for themanufacture of a rabies vaccine, according to which:

-   -   a) a batch of cells is produced,    -   b) the batch of cells is infected with rabies virus,    -   c) the rabies virus is purified from the infected-cell culture        supernatant that is recovered from the infected batch of cells        according to a method of the invention,    -   d) the suspension of purified virus is mixed in a storage        buffer, and    -   e) the suspension of purified virus obtained in c) is divided up        in the form of single-dose or multidose vaccines.

According to one embodiment of the vaccine manufacturing process, thestorage buffer is a lyophilization buffer. In this case, the suspensionof purified virus is mixed in the lyophilization buffer, the mixture isthen divided up in the form of single-doses or multidose vaccines, andfinally the vaccine doses are lyophilized.

In a preferred embodiment of the vaccine manufacturing process, all thesteps of the process are carried out with exogenous products ofnon-animal origin. The vaccine is free of any exogenous product ofanimal origin, which increases the quality of the vaccine obtained interms of biological safety.

Finally, a subject of the invention is a vaccine containing purifiedrabies virus according to which the amount of residual DNA measured byquantitative PCR and the amount of total proteins which are present inone effective dose of vaccine are less than 20 pg of residual DNA andless than 40 μg of total proteins. Preferably, one effective dosecontains less than 10 pg of residual DNA and less than 20 μg of totalproteins. More preferably, at least 70% of the total proteins are rabiesproteins. Ultimately, the vaccine is free of any exogenous product ofanimal origin. The rabies virus contained in the vaccine may beinactivated or attenuated. The densitometric analysis of theelectrophoretic profile of a vaccine sample, obtained afterpolyacrylamide gel electrophoresis and visualization with coomassieblue, in fact shows that more than 70% of the proteins are of viralorigin. The five rabies proteins, i.e. envelop glycoprotein G,nucleoprotein N, Phosphoprotein P, matrix protein M, and RNA dependantRNA polymerase L are essentially found onto a SDS-PAGE (Sodium DodecylSulfate-Polyacrylamide gel electrophoresis) carried out in the presenceof 2-mercaptoethanol. Furthermore, the particle size analysis of avaccine sample by means of the zetasizer Nano ZS machine (MalvernInstruments), which measures the Brownian motion of the particles on thebasis of “quasielastic” light scattering (Dynamic Light scattering),shows the existence of a single population of particles of between 100and 300 nm with an average value at 180 nm, which corresponds to theaverage size of the rabies virus. The analysis of a vaccine sample byelectron microscopy analysis shows the presence of viral particleshaving the classical bullet shape of the rabies virus. The vaccineaccording to the invention is therefore in the form of a homogeneoussuspension of whole purified rabies viruses wherein the particle sizeanalysis by means of the Zetasizer Nano ZS machine shows the existenceof a single peak between 100 and 300 nm approximately. This homogeneoussuspension of purified rabies viruses with the purity characteristics asdescribed can be obtained by implementing the purification method asdescribed in the invention.

The official test accepted by the WHO for evaluating the effectivenessof a rabies vaccine is described in the WHO Rabies monograph (WHOTechnical Series Report 941-January 2007 and European Pharmacopeiamonograph 0216 (“Rabies Vaccine for Human Use Prepared in CellCultures”). In order to be effective, one vaccine dose should contain atleast 2.5 IU determined according to European Pharmacopeia monograph0216.

The potency of rabies vaccine according to European Pharmacopeiamonograph 0216 is determined by comparing the dose necessary to protectmice against the effects of a lethal dose of rabies virus, administeredintracerebrally, with the quantity of a reference preparation of rabiesvaccine necessary to provide the same protection. For this comparison areference preparation of rabies vaccine, calibrated in InternationalUnits, and a suitable preparation of rabies virus for use as thechallenge preparation are necessary.

The International Unit is the activity contained in a stated quantity ofthe International Standard. The equivalence in International Units ofthe International Standard is stated by the World Health Organisation.

The test described below uses a parallel-line model with at least 3points for the vaccine to be examined and the reference preparation.Once the analyst has experience with the method for a given vaccine, itis possible to carry out a simplified test using a single dilution ofthe vaccine to be examined. Such a test enables the analyst to determinethat the vaccine has a potency significantly higher than the requiredminimum, but does not give full information on the validity of eachindividual potency determination. The use of a single dilution allows aconsiderable reduction in the number of animals required for the testand must be considered by each laboratory in accordance with theprovisions of the European Convention for the Protection of VertebrateAnimals used for Experimental and other Scientific Purposes.

-   -   Selection and distribution of the test animals. Use healthy        female mice, about 4 weeks old, each weighing 11-15 g, and from        the same stock. Distribute the mice into 6 groups of a size        suitable to meet the requirements for validity of the test and,        for titration of the challenge suspension, 4 groups of 5.    -   Preparation of the Challenge Suspension. Inoculate mice        intracerebrally with the Challenge Virus Standard (CVS) strain        of rabies virus and when the mice show signs of rabies, but        before they die, euthanise them, then remove the brains and        prepare a homogenate of the brain tissue in a suitable diluent.        Separate gross particulate matter by centrifugation and use the        supernatant liquid as the challenge suspension. Distribute the        suspension in small volumes in ampoules, seal and store at a        temperature below −60° C. Thaw one ampoule of the suspension and        make serial dilutions in a suitable diluent. Allocate each        dilution to a group of 5 mice and inject intracerebrally into        each mouse 0.03 ml of the dilution allocated to its group.        Observe the mice for 14 days. Calculate the LD50 of the        undiluted suspension using the number in each group that,        between the 5th and 14th days, die or develop signs of rabies.    -   Determination of potency of the vaccine. Prepare 3 fivefold        serial dilutions of the vaccine to be examined and 3 fivefold        serial dilutions of the reference preparation. Prepare the        dilutions such that the most concentrated suspensions may be        expected to protect more than 50 percent of the animals to which        they are administered and the least concentrated suspensions may        be expected to protect less than 50 percent of the animals to        which they are administered. Allocate the 6 dilutions, 1 to each        of the 6 groups of mice, and inject by the intraperitoneal route        into each mouse 0.5 ml of the dilution allocated to its group.        After 7 days, prepare 3 identical dilutions of the vaccine to be        examined and of the reference preparation and repeat the        injections. 7 days after the second injection, prepare a        suspension of the challenge virus such that, on the basis of the        preliminary titration, 0.03 ml contains about 50 LD50. Inject        intracerebrally into each vaccinated mouse 0.03 ml of this        suspension. Prepare 3 suitable serial dilutions of the challenge        suspension. Allocate the challenge suspension and the 3        dilutions, 1 to each of the 4 groups of 5 control mice, and        inject intracerebrally into each mouse 0.03 ml of the suspension        or dilution allocated to its group. Observe the animals in each        group for 14 days and record the number in each group that die        or show signs of rabies in the period 5-14 days after challenge.        The test is not valid unless:    -   for both the vaccine to be examined and the reference        preparation the 50 percent protective dose lies between the        largest and smallest doses given to the mice;    -   the titration of the challenge suspension shows that 0.03 ml of        the suspension contained not less than 10 LD50;    -   the statistical analysis shows a significant slope and no        significant deviations from linearity or parallelism of the        dose-response curves;    -   the confidence limits (P=0.95) are not less than 25 percent and        not more than 400 percent of the estimated potency.        The vaccine complies with the test if the estimated potency is        not less than 2.5 IU per human dose.

The administration of one effective dose of a rabies vaccine (i.e. atleast 2.5 IU) intramuscularly to humans according to the vaccination orsero-vaccination protocols normally recommended induces the developmentof protective immunity. Moreover, it has been observed that, when theamount of rabies virus contained in one vaccine dose according to theinvention is at least 4.5 IU on the basis of the measurement ofglycoprotein G by ELISA, this amount corresponds to at least oneeffective dose of vaccine (i.e. contains at least 2.5 IU according tothe NIH test).

The effective dose of the vaccine, which is injected intramuscularly inhumans, is generally contained in a volume of 0.5 to 1 ml of a liquidsuspension, which is ready to use or obtained by simple thawing or elseobtained extemporaneously by reconstituting a lyophilisate with asolvent.

The vaccination protocols used to protect humans against rabies are wellknown and differ depending on whether a preventive or curativevaccination is involved. Usually, in the case of a preventivevaccination, the prime vaccination protocol comprises two or 3intramuscular injections of one effective dose of vaccine. Thevaccination boosters are then given, at regular intervals, byadministering a single effective dose of vaccine. In the case of acurative vaccination, the vaccination protocols differ depending onwhether or not the individual exposed to the rabies virus has alreadybeen vaccinated and depending on the country. The vaccination protocolnormally recommended in nonimmunized or poorly immunized individuals, inparallel with the administration of an antirabies serum, comprises 5successive intramuscular injections of one effective dose of vaccineover a period of 1 month, followed by a booster at 3 months. The numberof injections is reduced to 3, or even to one, when the individualsexposed to the virus have previously received a complete preventivevaccination. Other vaccination protocols in the context of curativevaccination in nonimmunized or poorly immunized individuals that make itpossible to reduce the number of injections and/or amount of vaccineantigen administered can be used in the context of the presentinvention. These are in particular the “Zagreb” protocol, whichcomprises 4 intramuscular injections of one effective dose of vaccine(with 2 injections at different sites being given at day D0, followed byan injection at D7 and then at D21), or intradermal vaccinationprotocols.

The present invention will be understood more clearly in light of thefollowing examples which serve to illustrate the invention without,however, limiting the content thereof.

Example 1 Comparison of Various Chromatographic Supports in the Methodfor Purifying the Rabies Virus 1-1) Production of the Rabies Virus onVERO Cells in Serum-Free Medium

Cells of the VERO line, after having been adapted to culture conditionsin serum-free medium as described in WO 01/40443, were transferred intoa 10- to 20-liter biogenerator containing cytodex 1 microcarriers in VPSFM medium (Invitrogen). After a culture period of 3 to 4 days at 37° C.with the pH being maintained at approximately 7.2±0.2, with the oxygensaturation being maintained at 25%±10% and with the medium beingsubjected to slight stirring, the cells were infected with rabies virusat a multiplicity of infection of 0.01 in a viral infection mediumcontaining VP SFM medium (Invitrogen). In the case of the tests whichwere carried out with the Fractogel EMD-SO₃ ⁻ chromatographic support,the rabies virus production was carried out on a scale of 150 liters,using 150-liter biogenerators. The infected-cell culture supernatantswere harvested at days D7 (H1), D11 (H2) and D15 (H3). After eachharvest, new viral infection medium was reintroduced.

1.2) Clarification and Analysis of Harvests

The clarification step was carried out using two successive frontalfiltrations; the first using an 8 μm polypropylene pre-filter (SartopurePP2, SARTORIUS) which removes the few microcarriers drawn up during theharvesting, the Vero cells detached from the supports and the large celldebris; the second using an PES filter, composed of the combination oftwo filters, 0.8 μm and 0.45 μm (Sartopore 2, SARTORIUS), which removesthe aggregates.

The amount of rabies virus present in the clarified harvests wasdetermined by measuring the amount de glycoprotein G (gpG) measured bythe following ELISA method:

Approximately 0.12 μg/100 μl of a solution of an anti-gpG monoclonalantibody 1112-1 (the characteristics of which are described in Journalof Clinical investigation (1989), volume 84, pages 971 to 975),prediluted in a coating buffer (0.2 M carbonate/bicarbonate buffer, pH9.6), were dispensed into the wells of an ELISA microplate. Afterincubation overnight in a cold room, followed by several washes in awashing buffer (phosphate buffer supplemented with 0.05% Tween 20), 100μl of a saturation buffer (phosphate buffer supplemented with 1% bovineserum albumin) were dispensed into each well. After incubation for onehour at 37° C., followed by several washes, a dilution range of eachtest sample was prepared in a dilution buffer (phosphate buffersupplemented with 0.05% Tween 20 and 0.1% serum albumin). In parallel, adilution range of a reference standard, which was calibrated withrespect to the international reference of the NIBSC (for example,PISRAV), was prepared in each microplate. After a further incubation forone hour at 37° C., followed by several washes, 100 μl of a solution ofan anti-gpG mouse monoclonal antibody D1 (the characteristics of whichare described in Biologicals (2003), volume 31, pages 9 to 16), whichwas biotinylated and used after dilution to 1/5000 in the dilutionbuffer, were dispensed into each well. The plates were left for 1 hourat 37° C. and then washed several times before dispensing 100 μl of asolution of streptavidin coupled to peroxydase (Southern BiotechnologyAssociates), prediluted to 1/15000 in the dilution buffer, into each ofthe wells. After a further incubation for one hour at 37° C., followedby several washes, 100 μl of a solution of 0.05 M citrate buffer, pH 5,containing the revealing substrate (O-phenylenediamine), were dispensedinto each well. After an incubation time of 30 minutes at ambienttemperature in the dark, the revealing reaction was stopped by adding 50μl/well of a 2N solution of H₂SO₄. The spectrophotometric reading of themicroplates was carried out at two wavelengths (492 nm and 620 nm). Theoptical density measured is the difference between the two readings soas to take into account the absorption by the plastic. The relativeactivity was calculated by the parallel lines method according to therecommendations of the European Pharmacopeia. The rabies virus titer ofthe sample is based on the determination of the concentration of rabiesvirus glycoprotein G, which is expressed in IU/ml relative to thereference.

The amount of total proteins present in the clarified harvests wasmeasured using the conventional Bradford method sold in the form of akit by Biorad (ref: 500-0006).

The amount of DNA present in the clarified harvests was measured byqPCR. The working protocol is similar to that which is described byLebron J. A. et al., in Developments in Biologicals (2006), vol 123, pp.35-44. After having extracted the residual DNA from the clarifiedharvests by means of the commercial Extractor kit from Wako PureChemicals, a fixed amount of an exogenous DNA, which serves as aninternal control for the PCR amplification, was introduced into eachsample. In parallel, a sample of genomic DNA originating from Vero cellslysed by successive freezings/thawings, subsequently treated with RNaseA, in a proportion of 2 mg of RNase A per 2.5×10⁵ cells, was preparedand then, finally, subsequently purified using the QIAamp Virus BioRobot9604 kit (QIAGEN). The purified DNA was quantified by spectrophotometryat 260 nm. Using this purified DNA (standard DNA), a calibration rangewas prepared by performing 10-fold dilutions. The samples originatingfrom the clarified harvests and also the samples of the calibrationrange were subsequently subjected to a cycle of PCR amplification, afterhaving added, for each test, the fluorescent probe Alpha-MPH3, the twoprimers and the 2× QuantiTect Probe Master Mix premix (Qiagen) in avolume of completely nuclease-free water (qs 50 μl). The amplificationcycle was carried out using the Light Cycler 480 machine (Roche AppliedScience) using the program: 95° C., 15 min; 40 cycles comprising twosteps 95° C., 15 sec; 60° C., 60 sec. The amount of residual DNAextracted from the clarified harvests was then calculated, byinterpolation, on the basis of the measurement of the fluorescenceobserved compared with the calibration range established with thestandard DNA. The amount of residual DNA was adjusted by a correctivefactor corresponding to the charge efficiency measured for the sample bymeans of the quantification of the exogenous DNA. In general, theconcentrations of glycoprotein G (corresponding to the rabies virustiter), of total proteins and of DNA in the clarified harvests were inranges of from 1 to 3 IU/ml for the glycoprotein G, from 50 to 100 μg/mlfor the total proteins and from 5 to 50 ng/ml for the DNA.

1.3) Measurement of the Performance Levels of Various ChromatographicSupports in Terms of their Ability to Remove Nucleic Acids and to Retainthe Rabies Virus

The elimination of the cellular DNA in the clarified harvests wasevaluated on the following chromatographic supports:

Anionic Supports

-   -   Fractogel® EMD TMAE (Merck) (strong anion exchanger);    -   Fractogel EMD DEAE (Merck) (weak anion exchanger);    -   Sartobind® Q positively charged membrane (Sartorius);    -   Mustang® Q positively charged membrane (Pall).

Cationic Support

-   -   Fractogel® EMD SO₃ ⁻ (Merck) (strong cation exchanger).

Preliminary tests were carried out on the Sartobind® positively chargedmembrane so as to identify the best salt concentration for eluting thevirus by anion exchange chromatography. These tests showed that thevirus was eluted throughout the salt gradient, including at 1M. This wasalso true for the other anionic supports that were tested. The elutionprofiles obtained led us to choose a strong concentration of NaCl foreluting the maximum of virus. Since DNA began to be eluted when the NaClconcentration was around 350-400 mM, the compromise was to choose a NaClconcentration of 450 mM.

The anionic supports were subsequently equilibrated in 20 mM Tris buffercontaining 150 mM NaCl, pH 7.5. The clarified harvests were then broughtinto contact with the various supports studied. The anionic supportswere subsequently rinsed in 20 mM Tris buffer containing 150 mM NaCl, pH7.5. The virus was subsequently detached from the support by means of a20 mM Tris elution buffer containing 450 mM Nacl, pH 7.5.

The tests on a Fractogel column were carried out using the clarifiedharvest H1. Between 35 and 50 IU of rabies virus and between 13 and 25μg of DNA were injected per ml of gel.

The tests on membrane were carried out using the clarified harvest H2.13 IU of rabies virus and between 350 and 450 ng of DNA were injectedper cm² of membrane.

The working conditions used for the cation exchange chromatography onFractogel® EMD SO₃ ⁻ Support were also optimized. The Fractogel® EMD SO₃⁻ support was equilibrated using a 20 mM Tris buffer containing 150 mMNaCl, pH=7.5. The tests on Fractogel® EMD SO3⁻ support were carried outusing the clarified harvest H1. The conductivity of the medium (between14 mS/cm and 18 mS/cm) and the pH (between 7.5 and 7.7) were verified.Approximately 50 IU (on the basis of the assaying of glycoprotein G) ofrabies virus were injected per ml of gel. The filtrate was collected toassess the amount of virus which was not bound to the support; thesupport was subsequently washed in 20 mM Tris buffer containing 150 mMNaCl, pH=7.5. The virus was next eluted in 20 mM Tris buffer containing600 mM NaCl, pH=7.5, with recovery of the viral peak in an independentfraction. The support was finally regenerated in 20 mM Tris buffercontaining 1M NaCl, pH=7.5.

The results obtained are given in table II below.

TABLE II Performance levels of the various chromatographic supportsCharacteristics Amount of the Residual DNA chromatographic Virus DNAtiter removed support yield (ng/ml) (as %). Mustang Q 47%  4** 93%Sartobind Q 50%  1** 98% Fractogel 54% 196** 48% EMD-TMAE Fractogel 70%161** 73% EMD-DEAE Fractogel 89%    4.15***  99.7%*** EMD-SO3 *therabies virus titer was determined on the basis of the measurement ofglycoprotein G by ELISA **the residual DNA titer was measured by qPCRdirectly on the product recovered ***the residual DNA titer was measuredby qPCR after concentration of the eluate by a factor of 6 byultrafiltration.

The positively charged membrane supports (Sartobind® (Sartorius) orMustang® Q (Pall)) facilitate the removal of the DNA (more than 90% ofthe DNA is removed in the fraction collected), but on the other hand,the harvested virus yields are relatively low (≦50%). With regard to thesupports based on positively charged gels (Fractogel® EMD TMAE (Merck),Fractogel® EMD DEAE (Merck)), it is rather the opposite tendency that isobserved, i.e. a better harvested virus yield (≧50%) but to thedetriment of the removal of the DNA (73% of the DNA is removed in thebest of cases). On the other hand, when the Fractogel® EMD SO₃ ⁻cationic support is used, both a very good DNA removal (more than 95% ofthe DNA is removed in the fraction collected) and a very good harvestedvirus yield (≧70%) are observed. The chromatographic performance levelsof the Fractogel® EMD SO₃ ⁻ cationic support are therefore better thanthose that are observed with the anionic chromatographic supports.

1-4) Measurement of the Chromatographic Performance Levels of VariousCationic Chromatographic Supports

The previous results having shown that a strong cation exchanger supportis better than an anion exchanger support we sought to define thefeatures of the cation exchanger chromatographic support which give thebest harvested virus yields. The characteristics of the strong cationicsupports which were tested were the following:

-   -   Sartobind® S negatively charged membrane (Sartorius): a        cellulosic membrane onto which sulfonic acid groups (ligands)        are grafted;    -   Mustang™ S negatively charged membrane (Pall): polyethersulfone        membrane onto which sulfonic acid groups (ligands) are grafted;    -   Capto™ S Gel (GE Healthcare): gel of which the matrix is based        on agarose, onto which sulfoethyl groups are grafted through        dextran spacers;    -   SP Sepharose XL gel (GE Healthcare): gel of which the matrix is        based on agarose onto which sulfopropyl groups (ligands) are        grafted through dextran spacers;    -   Toyopearl® SP 650 (Tosoh): gel of which the matrix is based on        polymethacrylate onto which sulfopropyl groups (ligands) are        grafted;    -   Fractogel® EMD SO₃ ⁻ (Merck): gel of which the matrix is based        on polymethacrylate, onto which sulfoisobutyl groups (ligands)        are grafted through flexible spacers made of polymeric chains        obtained by polymerization of a monomer having the following        formula: CH₂═CH—CO—NH—C(CH₃)₂—CH₂-SO₃ ⁻        Gels of which ligands are grafted to the matrix through spacers        have a “tentacle” structure that may facilitate the binding of        the protein onto the ligand.

Various parameters were evaluated for the chromatography on Sartobind® Smembrane (Sartorius) (support volume: 7 ml): the pH of the clarifiedharvest injected, which was in a range of from 7.5 to 8.0, theconductivity of the clarified harvest injected, which was in a range offrom 4.5 mS/cm to 18 mS/cm, and the viral load injected, which was in arange of from 4 IU/ml to 11 IU/ml of support. Whatever the parametersstudied the virus yields obtained were always ≦35%.

In the case of the chromatography on Mustang™ membrane S (Pall) (supportvolume: 10 ml), the pH and the conductivity of the clarified harvestwere, respectively, 7.5 and 14 mS/cm, while the viral load injected was32 IU/ml of support. The virus yield obtained was very low (≦10%).

The protocol that was used for the chromatography on Fractogel® EMD SO₃⁻ as described in paragraph 1.3 was applied for the other gelchromatography supports. The only distinction is about the regenerationof the Toyopearl® SP 650 gel that was made in Tris 20 mM buffercontaining NaCl 2M, pH=7.5. The same pH and conductivity conditions asthose of the chromatography on Fractogel® EMD SO₃ ⁻ were used. Exceptfor the chromatography carried out on a Fractogel® EMD SO₃ ⁻ supportwhere the amount of virus found in the filtrate was about 5% of thetotal amount of virus injected in the chromatography column, the amountsof virus found in the filtrates of the other chromatography carried outon the other 3 supports (Capto™ S, SP Sepharose XL and Toyopearl® SP650) were between 80 and 99% of the total amount injected. Accordingly,the harvested virus yields were very low (<10%).

Therefore, the structure of the ligands as well as the matrix play animportant role since among all the strong cation exchangerchromatographic supports tested only the chromatographic support withsulfoisobutyl ligands grafted onto a polymethacrylate matrix gave goodresults.

In conclusion, out of all the anionic and cationic chromatographicsupports tested, only a tentacle support comprising a polymethacrylatematrix onto which sulfoisobutyl groups are grafted like the support soldby Merck under the commercial name Fractogel®EMD SO₃ ⁻ exhibitsperformance levels that are really exploitable from an industrial pointof view, since the virus yield is >70% and removes more than 95% of theDNA in a single step.

Example 2 Advantage of the Combination of a Step of Chromatography onFractogel® EMD SO₃ ⁻ Support with a Benzonase Treatment Step in theMethod for Purifying the Rabies Virus

In order to evaluate the advantage of this combination, this method wascompared with a method for purifying the rabies virus by doublebenzonase treatment. Benzonase treatment is conventionally used toremove nucleic acids that are contained in a biological product. Whenthis enzymatic treatment is repeated, the removal of the DNA is furtherincreased.

A method for purifying the rabies virus which uses a “double benzonasetreatment” was compared with the protocol of the invention, according towhich a step of cation exchange chromatography on Fractogel® EMD SO₃ ⁻support was combined with a benzonase treatment.

In the case of the double benzonase treatment (UF-Bz-UF-Bz), thepurification protocol that was used corresponds to that which isdescribed in Table III below:

TABLE III Purification of the rabies virus by double benzonase treatment(UF—Bz—UF—Bz).

In the case of the treatment combining cation exchange chromatography onFractogel® EMD SO₃ ⁻ support and a benzonase treatment (CEX-UF-Bz), thepurification method that was used corresponds to that which is describedin Table IV below:

TABLE IV Purification of the rabies virus by combination of cationexchange chromatography on Fractogel ® EMD SO₃ ⁻support and benzonasetreatment (CEX-UF-Bz).

In both cases, the purification method was carried out starting from thesame clarified harvest H1 having a volume of approximately 20 literswhich was divided up into two equal parts.

In the case of the UF-Bz-UF-Bz method, the two concentrations werecarried out on PES Medium Screen 1001(D membrane (PALL) combined with adiafiltration into 20 mM Tris buffer containing 150 mM NaCl, pH=7.5 byultrafiltration, the first ultrafiltration step resulting in reductionof the clarified harvest volume by a factor of approximately 20, whilethe second ultrafiltration led to reduction of the clarified harvestvolume by an overall factor of approximately 100. Before each benzonasetreatment, a solution of MgCl₂ was added such that the concentration inthe retentate was 2 mM. The benzonase treatment was carried out byadding 15 U/ml of crude harvest to the reaction medium and leaving thereaction medium for 2 hours at laboratory temperature.

In the case of the CEX-UF-Bz method, the chromatography step was carriedout according to the methods described in paragraph 1-3. The eluatecontaining the purified virus was subsequently concentrated by a factorof approximately 5 and diafiltered and then benzonase-treated using thesame protocol as that which was used in the UF-Bz-UF-Bz method.

In both methods, the ultracentrifugation step was carried out on 34-60%sucrose cushions with a 45 Ti type rotor at 21000 rpm for 2 h at +5° C.The fractions of the gradient containing the purified virus wererecovered, combined, and then analyzed in terms of their DNA, virus andtotal protein content.

Table V below indicates, at the various stages of purification, the DNA,gpG and total-protein titers obtained as a function of the method used.

TABLE V Assessment of the two purification methods PurificationUF-Bz-UF-Bz CEX-UF-Bz stage Parameters method method Clarified volume9620 ml 9620 ml harvest DNA titer (ng/ml) 270   gpG titer (IU/ml) 1.7DNA/gpG 713 × 10³pg/4.5 IU UF-Bz-UF volume 100 ml DNA titer (pg/ml) NTgpG titer (IU/ml) 106.6 IU/ml CEX-UF volume 100 ml DNA titer (pg/ml) NTgpG titer (IU/ml) 113 IU/ml After UC volume 800 ml 800 ml DNA titer(pg/ml) 1.1 × 10³ <0.1  gpG titer (IU/ml) 9.7  8.46 Total proteins/gpG36 μg/4.5 IU 24 μg/4.5 IU DNA/gpG titer 510 pg/4.5 IU <50 pg/4.5 IULog₁₀ reduction total DNA 3.47 >4.5  Virus yield* 63% 55% UF/Bz/UF:corresponds to the stage after the second ultrafiltration step but justbefore the second benzonase treatment in the UF-Bz-UF-Bz protocol.CEX/UF: corresponds to the stage after the first ultafiltration step butjust before the benzonase treatment in the CEX-UF-Bz protocol After UC:corresponds to the stage where the fractions of the gradient containingthe purified virus have been combined after ultracentrifugation andafter the total volume has been adjusted such as that it is 12.5 timesmore concentrated than the volume of the clarified harvest. *the virusyield is calculated on the basis of the gpG ELISA titers.

The results in table V show that the combination of chromatography onFractogel® EMD SO₃ ⁻ support followed by benzonase treatment (CEX-UF-Bzmethod) is much more effective in removing the DNA than a doublebenzonase treatment (UF-Bz-UF-Bz method). It is possible to reduce theamount of residual DNA by least a further 1 Log₁₀ using the CEX-UF-Bzmethod. These results were also confirmed on different volume scales.

It is also noted that the CEX-UF-Bz method also removes the contaminatedproteins more effectively than the UF-Bz-UF-Bz method, since there wasalmost half the total proteins per unit of virus (expressed in the formof IU of gpG) (5.4 μg in the CEX-UF-Bz method instead of 8 μg per IU ofgpG in the UF-Bz-UF-Bz method. This results from the fact that more than65% of the proteins are removed during the chromatography step onFractogel® EMD SO₃ ⁻ support. These results show that the combination ofa chromatography step on Fractogel® EMD SO₃ ⁻ support with a benzonasetreatment step in the method for purifying the rabies virus exerts acombined action on the removal of DNA in the clarified rabies virusharvests which is much greater than the effect observed during a doublebenzonase treatment.

1. A method of purifying rabies virus, the method comprising a singleion exchange chromatography purification wherein said ion exchangechromatography purification is a cation exchange chromatographypurification comprising a) applying a supernatant of a culture of cellsinfected with the rabies virus to a cation exchange chromatographysupport under conditions in which the rabies virus binds to the support,wherein the support comprises a polymethacrylate matrix onto whichsulfoisobutyl groups have been grafted by covalent bonding, and b)eluting the virus from the support.
 2. The method as claimed in claim 1,wherein the culture supernatant of cells infected with the rabies virusis free of animal serum or free of any serum protein.
 3. The method asclaimed in claim 2, wherein the culture supernatant of cells infectedwith the rabies virus is free of any exogenous protein of animal origin.4. The method as claimed in claim 3, wherein the culture supernatant ofthe cells infected with the rabies virus contains exogenous proteins ofnon animal origin at a concentration ≦15 mg/l.
 5. The method as claimedin claim 4, wherein the culture supernatant of cells infected with therabies virus is free of any exogenous product of animal origin.
 6. Themethod as claimed in claim 1, wherein the culture supernatant of cellsinfected with the rabies virus is a clarified supernatant.
 7. The methodas claimed in claim 1, wherein the amount of virus measured in theeluate corresponds to at least 70% and preferably to at least 80% of theamount of virus measured in the supernatant that was applied to thechromatographic support.
 8. The method as claimed in claim 7, whereinthe amount of total proteins measured in the eluate corresponds to lessthan 40% of the amount of total proteins measured in the supernatantthat was applied to the chromatographic support and the amount of DNAmeasured in the eluate corresponds to less than 5% of the amount of DNAmeasured in the supernatant that was applied to the chromatographicsupport.
 9. The method as claimed in claim 7, wherein the amount oftotal proteins measured in the eluate corresponds to less than 40% ofthe amount of total proteins measured in the supernatant that wasapplied to the chromatographic support and the amount of DNA measured inthe eluate corresponds to less than 1% of the amount of DNA measured inthe supernatant that was applied to the chromatographic support.
 10. Themethod as claimed in claim 1, wherein, further comprising treating theeluate, optionally concentrated, with a nuclease.
 11. The method asclaimed in claim 10, wherein the nuclease is an endonuclease.
 12. Themethod as claimed in claim 10, wherein the treated eluate is subjectedto ultracentrifugation on a sucrose gradient and recovering thefraction(s) of the gradient that contain(s) the purified virus.
 13. Themethod as claimed in claim 12, further comprising inactivating thepurified rabies virus with a viral inactivation agent.
 14. The method asclaimed in claim 13, wherein the viral inactivation agent isβ-propiolactone.
 15. The method as claimed in claim 13, wherein thepurification is conducted with products of non animal origin only.
 16. Amethod of manufacturing a rabies vaccine, the method comprising: a)infecting a culture of cells with the rabies virus, b) purifying therabies virus from an infected-cell culture supernatant according to themethod as claimed in claim 13, c) mixing the suspension obtained in b)with a storage buffer, and d) dividing the mixture obtained in c) into asingle-dose or multidose vaccine(s).
 17. A method for the manufacture ofa rabies vaccine, the method comprising: a) infecting a culture of cellswith the rabies virus, b) purifying the rabies virus from aninfected-cell culture supernatant according to the method as claimed inclaim 13, c) mixing the suspension of purified virus obtained in b) witha lyophilization buffer, d) dividing the mixture obtained in c) into asingle-dose or multidose vaccine(s), and e) lyophilizing the vaccinedose(s).
 18. A vaccine containing purified and inactivated rabies virus,wherein the amount of residual DNA measured by quantitative PCR and theamount of total protein present in one effective dose of vaccine or inone dose of vaccine which contains 2.5 IU as measured according to theEuopean Pharmacopeia monography, number 0216, are, respectively, lessthan 20 pg and less than 40 μg.
 19. The vaccine according to claim 18,wherein at least 70% of the total amount of proteins present in oneeffective dose of vaccine is rabies virus proteins.
 20. The vaccineaccording to claim 19, wherein the vaccine is free of any exogenousproduct of animal origin.